Chapter III Results
CHAPTER III
RESULTS
Gene and Protein Structure
The C. elegans genome contains a single gene (cosmid designation C40C9.5) encoding a homolog of vertebrate neuroligins. This gene, now designated nlg-1, spans approximately 5.7 kb of genomic sequence (Figure 8). The reading frame corresponding to the longest transcript (see below) encodes an 847 amino acid protein (including a predicted 17 amino acid N-terminal signal sequence). The structure of the predicted NLG-1 protein is similar to those of the mammalian neuroligins: a single-pass type I membrane protein with a large extracellular cholinesterase-like domain, and a small intracellular domain terminating in a PDZ binding motif (Figure 9). NLG-1 is 26-28% identical (45-47% similar) to the four human neuroligins and is most similar overall (28% identical, 47% similar) to human neuroligin-4 (Figure 7).
Through a combination of cDNA sequencing and RT-PCR analysis of transcripts, we documented several types of nlg-1 alternative splicing (Figure 8 and Figure 9). Exons 13 and 14 are variably present in nlg-1 transcripts; the skipping of these two exons occurs independently, and we have detected transcripts containing only exon 13, only exon 14, both exons, and neither exon. In addition, we have identified tandem alternative splice acceptor sites at the 5'-ends of exons 4 and 16, and a putative tandem alternative splice donor sites at the 3'-end of exon 14 (Figure 8 and Figure 9). If these splicing events are independent, there could be as many as twenty-four distinct NLG-1 isoforms.
Figure 7. Sequence alignments of the C. elegans NLG-1 and the hNLGN4 proteins
The sequences were aligned using the VNTI AlignX program. Identical amino acids are indicated with red letters on a yellow background, and structurally similar amino acids with black letters on a grey background. Predicted signal sequences are underlined in blue and transmembrane domains are double-underlined in black. Accession numbers: NLG-1: ACO52513; hNLGN4: NP_065793.
Figure 8. nlg-1 transcripts, deletion mutations and reporter constructs
Shown is the exon structure corresponding to the yk497a9 cDNA (GenBank accession FJ825295), augmented by the 5'- terminal 20 nucleotides and the 22-nucleotide SL1 sequence, which are from cDNA clone yk1657a10 (GenBank accession BJ767300). Blue regions are coding sequence; dark grey regions are untranslated regions. SL1 represents the trans-spliced leader sequence found at the 5'-end of many C. elegans mRNAs (Krause and Hirsh, 1987)). The vertical arrowheads above the exon diagram correspond to sites of alternative splicing; the white arrowheads indicate the variably present exons 13 and 14, and the black arrowheads indicate the documented tandem alternative splice acceptor sites at the 5'-ends of exons 4 and 16, and the putative tandem alternative splice donor sites at the 3'-end of exon 14. Also shown are the extents of the tm474 and ok259 deletions, the transcriptional reporter FRM77 and the translational reporter FRM253.
Cellular Expression and Sub-Cellular Localization of the NLG-1 Protein
A neuroligin transcriptional reporter is expressed in body-wall muscles and ~45 neurons
Identifying the cells that express neuroligin has several benefits. First, we can look for patterns in tissue type (e.g. neurons, muscles or gonad), transmitter type (e.g. GABA or ACh) or neuron type (sensory, motor, interneuron, etc.). Once the neuroligin expressing cells have been identified, we can use the known wiring diagram, behavioral circuitry and cell ablation data from the literature to interpret our behavioral data.
By comparing the genomes of three Caenorhabditis species (C. elegans, C. briggsae and C. remanei), we identified conserved sequence elements upstream of the nlg-1 coding region. We considered these conserved regions to be potential transcriptional regulatory elements. There are also conserved elements in the second intron of the nlg-1 gene, which we therefore included in our nlg-1 promoter constructs. Throughout, Pnlg-1 and the “nlg-1 promoter” refers to the 5’-end of the nlg-1 gene from 3563 base pairs upstream of the SL1 trans-splice site through the first 45 base pairs of exon 3.
We used a transgenic transcriptional reporter, with the nlg-1 promoter driving YFP expression (FRM77, Figure 8), to examine the cellular expression of nlg-1. We found that nlg-1 is expressed in a subset of neurons in C. elegans adults, including ~21 cells in the ventral nerve cord, four cells in the body and ~20 cells in the head (Figure 10, Figure 11 and Figure 12). We identified the nlg-1-expressing cells in the ventral nerve cord as the cholinergic VA and DA motor neurons (Figure 10, Figure 11 and Figure 12). We also identified the two AIY and two URB interneurons and the 4 URA motor neurons in the head, and the 2 PVD mechanosensory and 2 HSN motor neurons in the body, as nlg-1-expressing cells. Of these cells, the AIY interneurons are cholinergic (Altun-Gultekin et al., 2001), the PVD neurons are glutamatergic (Lee et al., 1999a), and the HSN neurons release both serotonin and acetylcholine (Desai et al., 1988, Duerr et al., 2001). Neurotransmitter assignments have not been reported for the remaining nlg-1-expressing neurons; however, they do not express GABAergic, dopaminergic, serotonergic, or glutamatergic reporters (see Methods). Finally, we also observed faint Pnlg-1::YFP expression in body wall muscles (Figure 11).
Figure 9. Structure of the C. elegans neuroligin protein (NLG-1)
Indicated are the positions corresponding to the ok259 and tm474 deletions, sites of alternative splicing, and the placement of YFP in functional fusion transgenes. Shown at higher resolution (below) is the C-terminal region of the protein (utilizing the single-letter amino acid code) from the transmembrane domain (TMD) to the terminal PDZ-binding motif. Pro-rich, proline rich region; N-glyco, putative N-linked glycosylation sites; O-glyco, region rich in putative O-linked glycosylation sites.
Figure 10. Cellular expression: nlg-1 transcriptional reporter
Confocal images of young adult transgenic animals (A-F) or L2 larval (G-I) animals expressing a Pnlg-1::YFP reporter (FRM77, see Figure 8 and Figure 9). Inset (G-I) is a more highly magnified view of the ventral nerve cord. The nlg-1 reporter is shown in green (A, B, D, E, G and H) and an eat-4 reporter (B and C; specific for glutamatergic neurons (Lee et al., 1999b)), unc-25 reporter (E and F; specific for GABA neurons (Eastman et al., 1999)) or unc-17 reporter (H and I; specific for cholinergic neurons (Alfonso et al., 1993, Duerr et al., 2008)) is shown in red. The Pnlg-1::YFP reporter is expressed in ~45 neurons in the head and body (out of the adult complement of 302 neurons). Anterior is to the left, ventral is down, and scale bar is ~10 μm.
Figure 11. Cellular expression: nlg-1 transcriptional reporter vs. ttx-3 transcriptional reporter
Confocal images of young adult transgenic animals expressing a Pnlg-1::YFP reporter (FRM77, see Figure 8 and Figure 9 An adult head view is shown in panels A, B and C. The nlg-1 reporter is shown in green and a ttx-3 reporter (specific for AIY neurons (Altun-Gultekin et al., 2001)) is shown in red (nr, nerve ring; vnc, ventral nerve cord). A section of the body-wall muscle is shown in panel D with higher magnification and higher gain). Anterior is to the left, ventral is down, and scale bars are ~10 μm. 
Figure 12. The relative position of nlg-1 positive cells with respect to cells of identified transmitter phenotype
The NLG-1 protein is localized to synaptic regions
To examine the subcellular localization of the NLG-1 protein, we generated a transgenic NLG-1::YFP fusion protein under control of the nlg-1 promoter (FRM253, Figure 8 and Figure 9). The diagrams in Figure 8 and Figure 9 show where the YFP sequence was inserted in the NLG-1::YFP fusion protein. We also generated NLG-1::YFP fusion proteins in which the YFP sequence was inserted at the N- or C-termini; these fusion proteins were either not efficiently synthesized or were unstable, and so we observe little or no expression. We believe that the fusion protein in FRM253 is functional because it rescues all nlg-1 mutant behaviors (see below). Confocal microscopy revealed that NLG-1::YFP is present at or near synapses (Figure 13 and Figure 14); localization in the synapse-rich nerve ring and ventral nerve cord is observed in embryos, and persists throughout development. NLG-1::YFP was also present in some neuronal cell bodies; this may reflect modest overexpression of the NLG-1::YFP transgene.
We compared the distribution of NLG-1::YFP with that of other synaptic proteins in the four sublateral nerve cords. White et al. reconstructed the nervous system from EM series, and found that in adult animals, each of these sublateral nerve cords contains 5 axons: SIA, SIB, SMB and SMD axons projecting from cell bodies in the head, and ALN/PLN axons projecting from cell bodies in the tail (White et al., 1986). These axons make periodic en passant neuromuscular synapses onto the adjacent body wall muscles. We determined that none of the cells with axons in the sublateral nerve cords expresses nlg-1, but the gene is expressed in the body wall muscles (Figure 11). We observed NLG-1::YFP-containing puncta along the sublateral nerve cords (Figure 14); these are of necessity muscle-derived, and are, therefore, postsynaptic. Furthermore, NLG-1::YFP puncta were apposed to presynaptic active zones (UNC-10/RIM-containing puncta) present in the axons (Figure 13 and Figure 14). We conclude that, at least in some cells, NLG-1::YFP is localized to postsynaptic regions.
Taken together, the similarity in amino acid sequence, overall protein structure and the sub-cellular localization of the protein argue strongly that the C. elegans NLG-1 is a bona fide neuroligin. This provides us with a system in which we can undertake structure-function studies.
Figure 13. A functional neuroligin YFP fusion protein is localized to synaptic regions in the nerve ring and nerve cords
Transgenic animals (expressing FRM253) were stained with anti-green fluorescent protein (GFP) (green; A and B) and anti-UNC-10/RIM (red; B and C). The head of a young adult hermaphrodite is shown. The positions of the nerve ring (nr), dorsal nerve cord (dnc) and ventral nerve cord (vnc) are indicated. Anterior is to the left and ventral is down. Scale bar ~10µm.
Figure 14. A functional neuroligin YFP fusion protein is localized to synaptic regions in the sublateral nerve cords
Transgenic animals (expressing FRM253) were stained with anti-green fluorescent protein (GFP) (green; A and B) and anti-UNC-10/RIM (red; B and C). Synaptic puncta in the sublateral nerve cord of a young adult hermaphrodite is shown. Anterior is to the left and ventral is down. Scale bar ~10µm.
Behavioral Phenotypes of Neuroligin Deficient (nlg-1) Mutants of C. elegans
We characterized the phenotypes associated with two independent nlg-1 mutations: ok259 and tm474. The nlg-1(ok259) mutation removes approximately half of the nlg-1 coding sequence (2,341 base pairs; Figure 8 and Figure 9) and is almost certainly a null mutation. The tm474 mutation is associated with a smaller (583-base pair) deletion, which removes exon 7 and part of exon 8 (Figure 8 and Figure 9).
Development, locomotion and nervous system structure are superficially normal in nlg-1 mutants
Animals homozygous for either of the nlg-1 alleles are viable and superficially wild type in their appearance, development and behavior. In addition, the nervous system of nlg-1 mutants is grossly normal: expression of neuronal reporters in live animals and indirect immunofluorescence for several different synaptic antigens in fixed specimens revealed no apparent difference between nlg-1(ok259) mutants and wild-type animals (data not shown).
We used two quantitative assays to measure general synaptic function in nlg-1 mutants. Response to the acetylcholinesterase inhibitor aldicarb is commonly used as a measure of cholinergic neurotransmission in C. elegans; resistance to aldicarb is associated with decreased ACh release (Miller et al., 1996). Locomotory behavior in swimming assays provides a general measure of motor neuron function and neurotransmitter release. We found that nlg-1 mutants did not differ from wild-type animals in their response to aldicarb in an acute response assay (data not shown).While aldicarb only affects ACh, mutants with aldicarb sensitivity or resistance have been shown to include mutants involving all neurotransmitter types and/or general synaptic activitiy (Miller et al., 1996, Sieburth et al., 2007). We also found that nlg-1 mutants were not appreciably deficient in swimming behavior (wild type = 153 ± 4 body bends/min; nlg-1(ok259) = 143 ± 11 body bends/min; details in Methods). We therefore conclude that elimination of nlg-1 function does not lead to dramatic deficits in synapse formation or function. However, as described below, we have identified a number of significant behavioral and biochemical differences between nlg-1 mutants and wild-type animals.
nlg-1 mutants lack a thermal response
When well-fed wild-type nematodes are placed in a thermal gradient, they preferentially accumulate at the temperature at which they were raised, presumably because this temperature is associated with food (Hedgecock and Russell, 1975). Wildtype animals avoid temperatures at which they have been starved. nlg-1 mutants, however, appear to lack a thermal response. They do not accumulate at a specific temperature, but instead, they move independently of ambient temperature (Figure 15). This atactic behavior was observed with both ok259 and tm474 homozygotes, and was independent of the temperature at which the animals were grown or their feeding state. Normal thermal responses were restored by transgenic expression of either NLG-1 or an NLG-1::YFP fusion protein (Figure 15). We conclude that nlg-1 mutants are either unable to sense temperature or are indifferent to changes in ambient temperature.
Figure 15. nlg-1 deletion mutants have thermotaxis defects
Animals were grown at 20°C and placed on a thermal gradient as described in Methods. Wild-type nematodes accumulated at their growth temperature. nlg-1 mutants did not accumulate at a specific temperature, but instead moved independently of temperature. Transgenic expression of a NLG-1::YFP fusion protein (FRM253) rescued the thermotaxis defect. Each data point represents the mean of six trials of 50 animals each ± standard deviation. At the 20°C temperature point, there is a statistically significant difference (P < 0.0001) between nlg-1 and wild type.
Spontaneous reversal frequency is reduced in nlg-1 mutants; this is a progressive phenotype
The most obvious behavioral output of the C. elegans nervous system is locomotion. C. elegans explores its environment using a directed chemotaxis and biased random locomotion. C. elegans moves on an agar plate by generating sinusoidal waves along the length of its body. When these waves are propagated from head to tail the animal moves forward, and when propagated from tail to head the animal moves backward (Gray and Lissmann, 1964). The transition from forward to backward movement is referred to as a "reversal." Such reversals can occur spontaneously (Pierce-Shimomura et al., 1999), or can be induced as a direct response to a sensory stimulus (Chalfie et al., 1985a, Bargmann, Mori and Ohshima, 1995).
We found that nlg-1 mutants move forward for a much longer time than wild-type animals before initiating backward movement (Figure 16). This decrease in spontaneous reversal rate was observed with both ok259 and tm474 homozygotes, and was rescued by transgenic expression of the NLG-1::YFP fusion protein (Figure 16). However, although the likelihood of a reversal event was greatly reduced in nlg-1 mutants, the duration of backing, once initiated, appeared to be normal (data not shown).
We also observed that the decrease in reversal likelihood was progressive: the decrease was significant in young larvae, but far more pronounced in adults (Figure 17). We note that other phenotypes of nlg-1 mutants, including insensitivity to temperature and lack of response to 1-octanol (discussed below), do not appear to be progressive. This suggests a role for NLG-1 in synaptic maintainance for certain synapses
Figure 16. nlg-1 deletion mutants have spontaneous reversal deficits
Animals were removed from food and monitored for spontaneous reversal of direction. Each animal was observed for three minutes; each data bar represents the mean of 25 animals ± standard deviation. The nlg-1 mutants moved forward for a much longer time before initiating backward movement. Transgenic expression of a NLG-1::YFP fusion protein (FRM253) rescued this mutant phenotype. (***) indicates a statistically significant difference (P<0.0001) between nlg-1 and wild-type animals.
Figure 17. Spontaneous reversals in larvae and adults: the nlg-1 spontaneous reversal deficit is progressive
Animals were removed from food and monitored for spontaneous reversal of direction. Each animal was observed for three minutes; each data bar represents the mean of 25 animals ± standard deviation. The nlg-1 mutants moved forward for a much longer time before initiating backward movement. This phenotype is progressive: 4-day-old (adult) animals have a more dramatic phenotype than 2-day-old (L3) animals.nlg-1 mutants have specific chemosensory deficits
Wild-type C. elegans hermaphrodites respond to a wide variety of volatile and water-soluble chemical cues; these responses have been described by Bargmann et al. (Bargmann et al., 1993, Bargmann) as well as others. In general, nlg-1 mutants responded normally to most attractants and repellants. However, we identified some specific chemosensory deficits in nlg-1 mutants. For example, nlg-1 mutants are not repelled by the normally aversive chemical 1-octanol (Figure 18).
The octanol-sensing deficit can not be ascribed to a general insensitivity to volatile chemicals (nlg-1 mutants respond normally to the volatile attractant diacetyl). It can also not be ascribed to an insensitivity to repellants in general (the mutants have a normal response to the repellant cupric acetate), or even to a general insensitivity to volatile repellants (the mutants have a normal response to the volatile repellant nonanone). The noxious 1-octanol response is mediated by the ASH neuron (Bargmann et al., 1993), but the worm responds normally to nose touch, which is also sensed by ASH (Kaplan and Horvitz, 1993). The behavior seems instead to be a specific lack of response to 1-octanol.
Figure 18. nlg-1 deletion mutants have octanol avoidance defects
Animals were grown at 20°C and placed on a chemotaxis gradient as described in Methods. Wild-type nematodes were averse to 1-octanol. In contrast, nlg-1 mutants were insensitive to octanol. Transgenic expression of a NLG-1::YFP fusion protein (FRM253) rescued this mutant phenotype. Each data point represents the mean of three trials of 25 animals each ± standard deviation. (***) indicates a statistically significant difference (P ≤ 0.0001) between nlg-1 and wild type.nlg-1 mutants have altered processing of conflicting chemosensory cues
In addition to defects in response to specific chemical cues, nlg-1 mutants also exhibit defects in the processing of two conflicting chemosensory inputs. For example, wild-type animals and nlg-1 mutants are comparably attracted to diacetyl and repelled by
cupric acetate (Figure 19). However, when animals are presented with these two compounds simultaneously (i.e., with a cupric acetate barrier between the animals and the attractant), nlg-1 mutants are far more likely than wild-type animals to cross the barrier in response to the attractant (Figure 19).
Figure 19. nlg-1 deletion mutants have Approach-Avoidance deficits
A chemo-attractant (2 µl of 0.1% diacetyl) was placed on one side of the plate, ~25 animals were placed on the opposing side of the plate and a barrier of Cu(CH3COO)2 was placed in the middle of the plate (see methods and panel A). The worms were allowed to move freely on the plate, and the worms that crossed the barrier were scored. All animals avoided the barrier when no attractant was presented (Cu(+) Diacetyl(-)), more than 90% of the animals accumulated at the attractant when no barrier was present (Cu(-) Diacetyl(+)) and when barrier and attractant were presented simultaneously (Cu(+) Diacetyl(+)), ~25 ± 2 % of wild-type (N2) animals and >60 ± 5 % of nlg-1 mutant animals crossed the barrier. For each genotype (N2 = wild-type, nlg-1 and rescue= Pnlg-1::NLG-1::YFP), three groups of 25 animals were assayed at 20°C as described in Methods. (*) indicates p<.01. All error bars are ± STD.nlg-1 Mutants Have Elevated Oxidative Stress
A completely unexpected result in the analysis of the nlg-1 mutant animals was the finding that the mutant animals had a shorter lifespan (Figure 21), increased sensitivity to oxidative stress (Figure 20) and metal stress (Figure 20 and Figure 22), and increased oxidative damage to their proteins (Figure 20). The nlg-1 mutants also have visible tissue damage, and a shorter time of optimal health as assessed by their swimming rates (Figure 40) and spontaneous locomotion (Figure 21).
Hypersensitivity to paraquat
The progressive nature of the spontaneous reversal phenotype in nlg-1 mutants suggested that the absence of neuroligin might trigger some type of degenerative process. We therefore evaluated the sensitivity of nlg-1 mutants to oxidative stress. Exposure to paraquat (N,N'-dimethyl-4,4'-bipyridinium dichloride) is commonly used as a paradigm for oxidative stress in C. elegans (Ishii et al., 1990). We found that nlg-1 mutants were significantly more sensitive to paraquat than wild-type animals, and this hypersensitivity was rescued by transgenic expression of a NLG-1::YFP fusion protein (Figure 20). In most organisms, including C. elegans, hypersensitivity to oxidative stress is often associated with decreased lifespan (Ishii et al., 1998, Senoo-Matsuda et al., 2001, Kondo et al., 2005), and this was true for nlg-1 mutants (Figure 21).
An oxidative damage biomarker is elevated in nlg-1 mutants
To examine oxidative damage directly, we measured protein carbonylation (an irreversible oxidative modification) in wild type, nlg-1 and mev-1 strains. We modified an approach used previously by Adachi, Yasuda and others to assess oxidative damage in C. elegans (Adachi et al., 1998, Yasuda et al., 1999), and developed an ELISA method (see Methods) to quantify oxidative damage. We included mev-1 mutants in our analysis because it was reported that mev-1 mutants are hypersensitive to paraquat (Ishii et al., 1990) and that they exhibit elevated levels of carbonylation. Paraquat significantly increased the protein carbonylation levels of all of the strains tested, although the levels remained much higher in nlg-1 mutants than in wild-type animals. Transgenic expression of the NLG-1::YFP fusion protein lowered protein carbonylation levels to those of wild type (Figure 20), confirming the specificity of this biochemical phenotype. Levels of carbonylated proteins in untreated and treated mev-1 mutants were comparable to those observed in similarly treated nlg-1 mutants. We conclude that, even in the absence of paraquat, nlg-1 mutants experience increased levels of oxidative stress.
nlg-1 mutants have reduced lifespan
Because nlg-1 mutants have increased sensitivity to oxidative stress and increased basal oxidative damage, we wanted to check whether they had altered lifespan under normal conditions. We found that nlg-1 mutants have significantly shorter lifespan than wild-type animals (Figure 21). Treatment with the antioxidant BHT improved lifespan of nlg-1 mutants, but any improvement in lifespan of wild-type animals was negligible (Figure 21). We also noted that the nlg-1 mutants stop spontaneous locomotion significantly sooner than wild-type animals.
Additional Stress Phenotypes of nlg-1 Mutants
We extended these studies by examining the toxic effects of mercury compounds, and we found that nlg-1 mutants were significantly more sensitive than wild-type animals to both inorganic (HgCl2) and organic (thimerosal) forms of mercury (Figure 20). The nlg-1 mutants were also more sensitive than wild-type animals to the toxic effects of copper (cupric acetate, Figure 22). However, there was no difference between the mutants and wild-type animals in their survival on cadmium (CdCl2, Figure 22); nlg-1 mutants are therefore not hypersensitive to all heavy metals.
nlg-1 mutants have normal sensitivity to thermal stress
Because nlg-1 mutants have increased sensitivity to oxidative stress, we tested whether they also exhibit increased sensitivity to thermal stress. We incubated worms at 34°C, and scored them for survival every 30 minutes. We found that nlg-1 mutant animals were no more sensitive to thermal stress than wild-type (N2) worms. nlg-1 mutant animals survived for 233 ± 53 minutes, while wild-type (N2) worms survived for 239 ± 50 minutes.
Analysis of tissue aging in nlg-1 deficient mutants
Garigan et al. (Garigan et al., 2002) reported that Nomarski differential interference contrast (DIC) microscopy provided an effective means of visualizing many features of tissue aging. They found that extensive tissue deterioration takes place during aging. Chow et al. reported that muscle deterioration in the pharynx could be seen as a function of age (Chow et al., 2006). Lippofuscin accumulation in the gut of the animal is another biomarker of aging (Pincus and Slack, 2010).
While examining nlg-1 mutant animals under high magnification, we observed accumulations of vacuole-like structures in the head regions of many adult animals (Figure 23). We do not yet know which tissue with which the vacuoles are associated. Observation of wild-type animals established that these vacuoles are a morphological feature of aging worms. We then quantified the number of vacuoles in wild-type and nlg-1 mutant animals at different ages. We found that vacuoles accumulate at a faster rate in nlg-1 mutants (Figure 23), and that nlg-1 mutants accumulate more vacuoles at the end of their lives (Figure 23) than wild-type animals, even though the mutants are chronologically younger. The observation of vacuoles is consistent with the results of the lifespan and healthspan assays, and taken together, suggest that nlg-1 mutants exhibit signs of accelerated aging.
Figure 21. nlg-1 mutants have reduced lifespan and healthspan
For each genotype (N2 and nlg-1), three groups of 25 animals were grown at 20°C as described in Methods, and assessed each day for survival and spontaneous movement; data points represent the mean percent survival ± standard deviation. The difference in mean lifespan between nlg-1 and wild type is statistically significant (Gehan-Breslow-Wilcoxon Test P < .0001). Treatment with antioxidants improved lifespan of nlg-1 mutants (Gehan-Breslow-Wilcoxon Test P < .0001), but improvement in lifespan of wild-type animals was negligible (Gehan-Breslow-Wilcoxon Test P = .4).
Figure 22. nlg-1 mutants are hypersensitive to copper, but not cadmium
Young adults (N2, nlg-1 mutants, and nlg-1 mutants expressing the FRM253 rescuing transgene) were transferred to plates containing either 0.7 mM cupric acetate (left panel) or 8.0 mM CdCl2 (right panel), and survival was scored every 24 hours. Each data point represents the mean of four trials (with at least 20 animals in each trial) ± standard deviation. The difference in mean survival times on cupric acetate between nlg-1 (1.8 ± 0.2 days) and wild type (4.2 ± 0.2 days) is statistically significant (P < .0001).
Figure 20. Neuroligin deficient mutants exhibit hypersensitivity to oxidative stress and elevated oxidative damage
A. Sensitivity to paraquat. Young adults were transferred to plates containing 1.5 mM paraquat and monitored daily for survival. Each data point represents the mean of six trials of 10 animals each ± standard deviation. The difference in mean survival times between nlg-1 (3.7 ± 0.6 days) and wild type (5.7 ± 0.4 days) is statistically significant (P < .0001). B. Sensitivity to thimerosal. Young adults were transferred to plates containing 77 nM thimerosal. Each data point represents the mean of three trials with at least 46 animals in each trial ± standard deviation. The difference in mean survival times between nlg-1 (1.3 ± 0.03 days) and wild type (3.0 ± 0.1 days) is statistically significant (P < .0001). C. nlg-1 mutants have elevated levels of oxidized proteins. Young adults were transferred to plates containing 1.8 mM paraquat (or control) and grown for 2 days, then assayed by ELISA for carbonyl modification of proteins. Values were normalized to protein concentration, and are presented relative to the value of wild type without paraquat (N2 = 2.43 ± 0.94 ng carbonyl/μg protein). Bars represent the means of four separate experiments ± standard deviation. "Rescue" = nlg-1 mutants expressing an integrated functional NLG-1::YFP transgene (FRM253). mev-1 mutants were previously shown to be hypersensitive to paraquat and to have elevated levels of oxidized protein (Ishii et al., 1990; Adachi et al., 1998). +Pq, -Pq represent growth with or without paraquat, respectively. The asterisk (*) indicates a statistically significant difference (P < .0001) between N2 with and without paraquat, the dagger (†) indicates a statistically significant difference (P = .0005) between nlg-1 without paraquat and wild type without paraquat, and the double dagger (‡) indicates a statistically significant difference (P = .0011) between mev-1 without paraquat and wild type without paraquat.
Figure 23. nlg-1 mutants show signs of premature aging
Nomarski images of the heads of 9-day old (left) and 12-day old (right) wild-type (top) and nlg-1 mutants (bottom). Arrows indicate some of the vacuoles counted. Below is a graph of the average number of vacuoles (± standard deviation) in the field when the grinder is in focus. Each bar represents data from 12 animals.Are Nematode and Mammalian Neuroligins Functionally Equivalent?
Based on the hypothesis that proteins with similar sequences, expression paterns and localization might play similar roles in synaptic processes, we inquired whether mammalian and nematode neuroligins were functionally equivalent. That is, can mammalian neuroligins substitute for NLG-1 in C. elegans? We therefore expressed the human NLGN4 and the rat Nlgn1 cDNAs (with a modified signal sequence and 3’-UTR, see Methods) under the control of the C. elegans nlg-1 promoter in nlg-1 null mutants and evaluated the resulting phenotypes.
Rescue of mutant behavioral phenotypes by transgenic expression of mammalian neuroligins
We found that transgenic expression of the modified hNLGN4 cDNA under the control of the C. elegans nlg-1 promoter rescued the spontaneous reversal phenotype of nlg-1 mutants (Figure 16 and Figure 24). In addition, the thermotaxis defect of nlg-1 mutants (Figure 15) was rescued by transgenic expression of either h NLGN4 or rNlgn1 cDNAs (Figure 25). Similarly, octanol avoidance was restored by transgenic expression of either hNLGN4 or rNlgn-1 cDNA (Figure 27), as was wild-type sensory integration in the Approach-Avoidance assay (Figure 26). Taken together, these results suggest that mammalian neuroligins can function at C. elegans synapses in a manner that is equivalent to C. elegans NLG-1, at least for these behaviors.
Figure 24. hNLGN4 rescues the nlg-1 spontaneous reversal phenotype
Animals were removed from food and monitored for spontaneous reversal. Each animal was observed for 3 minutes; each data bar represents the mean of 12 animals ± standard deviation. Transgenic expression of an hNLGN4 construct rescued the spontaneous reversal defect. (***) indicates a statistically significant difference (P ≤ .0001) between nlg-1 and wild-type (N2) or hNLGN4. N2 and hNLGN4 are not different.
Figure 25. hNLGN4 and rNlgn1 rescue the nlg-1 thermotaxis defect
Animals were grown at 20°C and placed on a thermal gradient as described in Methods. Wild-type (N2) nematodes accumulated at their growth temperature. Transgenic expression of mammalian neuroligin cDNAs rescued the nlg-1 mutant’s thermotaxis defect. Each data point represents the mean of six trials of ~50 animals each (± standard deviation).
Figure 26. hNLGN4 and rNlgn1 rescue the nlg-1 Approach-Avoidance defect.
Approach-Avoidance assays were performed as described in methods and Figure 19. Transgenic expression of an hNLGN4 or rNlgn1 construct rescued the nlg-1 mutant’s Approach-Avoidance defect. Each data point represents the mean of three trials of 25 animals each ± standard deviation. (***) indicates a statistically significant difference (P ≤ .0001) between nlg-1 and wild type, hNLGN4 or rNlgn1. hNLGN4 and rNlgn1 are not different than N2.Figure 27. hNLGN4 and rNlgn1 rescue the nlg-1 octanol chemotaxis deficit
Animals were grown at 20°C and placed on a chemotaxis gradient as described in Methods. Transgenic expression of an hNLGN4 or rNlgn1 construct promoter rescued the nlg-1 mutant’s chemotaxis defect. Each data point represents the mean of three trials of 25 animals each ± standard deviation. (***) indicates a statistically significant difference (P ≤ .0001) between nlg-1 and wild-type, hNLGN4 or rNlgn1. hNLGN4 and rNlgn1 are not different than N2.
Figure 28. Relative rescue for mammalian neuroligin transgenic animals
The results shown in the preceding figures were normalized by setting wild-type to 100% and nlg-1 to 0%, and then performing a linear interpolation between the two. hNLGN4 and rNlgn1 gave complete rescue for three of the four phenotypes measured. In the case of spontaneous reversal, we observed an increase in likelihood of reversal relative to wild type. We saw a similar increase when we overexpressed NLG-1 (not shown). We therefore believe that the elevated values for spontaneous reversal associated with the hNLGN4 and rNlgn1 transgenic strains are overexpression artifacts.Effects of ASD-related neuroligin mutations in C. elegans
We took a similar approach to investigate the importance of single amino acid substitutions on the function of C. elegans NLG-1 protein. In addition to frame-shift mutations (Jamain et al., 2003, Laumonnier et al., 2004), copy number variations, deletion mutations (Lawson-Yuen et al., 2008) and regulatory mutations (Daoud et al., 2009), several single amino acid substitutions in hNLGN4 and hNLGN3 have been shown to be associated with autism. These include hNLGN3 R451C (Jamain et al., 2003), hNLGN4 G99S, hNLGN4 K378R, hNLGN4 V403M, hNLGN4 R704C (Yan et al., 2005) and hNLGN4 R87W (Zhang et al., 2009) (Table 3). We identified the homologous amino acid in the C. elegans NLG-1 protein for four of these mutations (Table 4), and made the comparable mutations in the C. elegans nlg-1 cDNA. These constructs were derived from RM#980p (see Methods),
Table 4 Amino acid substitutions in hNLGN4 and hNLGN3 associated with autism, and the homologous amino acids in the C. elegans NLG-1 protein
which includes YFP in the coding sequence. We expressed these constructs under the control of the C. elegans nlg-1 promoter in nlg-1 null mutants, and examined the expression level and localization of the mutant proteins, as well as the extent of phenotypic rescue.
The four mutated proteins were trafficked properly and localized normally. This suggests that any mutant phenotypes observed arise from functional problems with the proteins, rather than deficits in trafficking. We found that the nlg-1 mutant’s decrease in spontaneous reversal rate (Figure 16) was fully rescued by transgenic expression of the V397M and R714C mutant constructs, whereas R430C and R62W constructs gave partial rescue for spontaneous reversal rate (Figure 29 and Figure 33). The nlg-1 mutant animal’s thermal insensitivity (Figure 15) was partially rescued, and some degree of thermal response was restored by transgenic expression of either the V397M or R714C constructs. The R430C and R62W mutant constructs, however, failed to rescue, and transgenic animals remained athermotactic (Figure 30 and Figure 33). Similarly, the aversive response to octanol was restored by transgenic expression of either the V397M or R714C construct (Figure 32 and Figure 33), as was wild-type sensory integration in the Approach-Avoidance assay (Figure 31 and Figure 33). R430C transgenic animals were insensitive to octanol, and sensory integration (in the Approach-Avoidance assay)
was significantly different from wild type, but not significantly different from nlg-1 mutant animals. R62W transgenic animals were slightly averse to octanol, and had impaired sensory integration; sensory integration (in the Approach-Avoidance assay) significantly different from wild-type, but not significantly different from nlg-1 mutant animals.
Taken together, these results suggest that V397M or R714C constructs can function in C. elegans in a way that is equivalent to wild-type C. elegans neuroligin, for these assays. The R430C and R62W mutations, however, appear to reduce NLG-1 function significantly but not completely.
Figure 29. ASD associated mutations: effects on spontaneous reversal
Animals were removed from food, and monitored for spontaneous reversal of direction. Each animal was observed for 3 minutes; each data bar represents the mean of 12 animals ± standard deviation. Transgenic expression of a R714C NLG-1::YFP or V397M NLG-1::YFP fusion protein rescued the nlg-1 mutant’s spontaneous reversal defect completely, while transgenic expression of a R430C NLG-1::YFP or R62W NLG-1::YFP fusion protein gave partial rescue. (***) indicates a statistically significant difference (P ≤ .0001) between nlg-1 and wild type and/or R430C and wild type. (**) indicates a statistically significant difference (P ≤ .001) between R62W and wild type.Figure 30 ASD associated mutations: effects on thermotaxis behavior
Animals were grown at 20°C, and placed on a thermal gradient as described in Methods. Transgenic expression of a R714C NLG-1::YFP or V397M NLG-1::YFP fusion protein rescued the nlg-1 mutant’s thermotaxis defect while transgenic expression of a R430C NLG-1::YFP or R62W NLG-1::YFP fusion protein did not. Each data point represents the mean of three trials of ~50 animals each ± standard deviation.
Figure 31. ASD associated mutations: effects on Approach-Avoidance
Approach-Avoidance assays were performed as described in Methods and Figure 19. Transgenic expression of R714C NLG-1::YFP or V397M NLG-1::YFP fusion proteins rescued the nlg-1 mutant’s Approach-Avoidance defect, while transgenic expression of a R430C NLG-1::YFP or R62W NLG-1::YFP fusion protein gave partial rescue. Each data point represents the mean of six trials of ~50 animals each ± standard deviation. (***) indicates a statistically significant difference (P ≤ .0001) between nlg-1 and wild type, R430C and wild type or R62W and wild type.
Figure 32. ASD associated mutations: effects on octanol chemotaxis
Animals were grown at 20°C, and placed on a chemotaxis gradient as described in Methods. Transgenic expression of a R714C NLG-1::YFP or V397M NLG- 1::YFP fusion protein rescued the nlg-1 mutant’s chemotaxis defect, while transgenic expression of a R430C NLG-1::YFP or R62W NLG-1::YFP fusion protein did not. Each data point represents the mean of six trials of ~50 animals each ± standard deviation. (***) indicates a statistically significant difference (P ≤ .0001) between nlg-1 and wild type, R430C and wild type or R62W and wild type.
Figure 33. Relative rescue of ASD-associated constructs
The results shown in the preceding figures were normalized by setting wild-type to 100% and nlg-1 to 0%, and then performing a linear interpolation between the two Transgenic expression of R714C NLG- 1::YFP or V397M NLG-1::YFP fusion proteins rescued all of the nlg-1 phenotypic defects, while expression of a R430C NLG-1::YFP or R62W NLG-1::YFP fusion protein gave partial or no rescue. In the case of spontaneous reversal, we observed an increase in likelihood of reversal relative to wild type. We saw a similar increase when we overexpressed NLG-1 (not shown). We therefore believe that the elevated values for spontaneous reversal associated with the transgenic strains are overexpression artifacts.
Analysis of Factors Required for C. elegans Neuroligin Localization and Function
Deletion constructs
In order to determine the contribution of various sections of the intracellular domain of the protein to neuroligin function, we evaluated a set of deletion constructs (Figure 34). These included deletions of the PDZ binding motif`, the constant region (exons 15-16), the variable region (exons 13-14) and the entire intracellular portion of the protein (exons 13-16). We expressed each of these constructs under the control of the C. elegans nlg-1 promoter in nlg-1 null mutants, and assessed neuroligin localization and function.
Figure 34 Schematic diagram of the neuroligin (NLG-1) deletion constructs generated and analyzed for this study.
With the exception of the deletion of the complete intracellular domain (exons 13‑16), each of the deletion constructs was robustly expressed, and fluorescence was observed in synapse-rich regions, including the dorsal, ventral and sublateral nerve cords, and the nerve ring. Within the dorsal and sublateral cords, which do not contain cell bodies, discrete puncta were observed; these puncta were indistinguishable from those observed in a control (wild-type) NLG-1::YFP strain. However, we only looked for the presence of discrete puncta, and did not stain with antibodies against other synaptic proteins or co-express the mutant NLG-1 proteins with other fluorescent synaptic fusion proteins to determine whether the localization of the mutant neuroligin is actually synaptic. Strains expressing the complete intracellular domain deletion construct (Δ 13-16) were much fainter than the control strain; however, puncta could still be observed, albeit with difficulty, in synaptic regions. Thus the intracellular domain does not appear to be necessary for the localization of NLG-1 in C. elegans.
We then examined the behaviors of these transgenic strains. We found that transgenic expression of the Δ 13-14 and Δ PDZ cDNAs under the control of the C. elegans nlg-1 promoter rescued the nlg-1 spontaneous reversal rate deficit (Figure 16), whereas Δ 13-16 and Δ 15-16 mutant constructs failed to rescue this phenotype (Figure 35). In addition, thermal responses were restored by transgenic expression of either the Δ 13-14 or Δ PDZ construct cDNAs under the control of the C. elegans nlg-1 promoter. The Δ 13-16 and Δ 15-16 mutant constructs, however, failed to rescue the thermal response, and transgenic animals remained athermotactic (Figure 36). Similarly, wild-type sensory integration in the Approach-Avoidance assay (Figure 37) was rescued by transgenic expression of either the Δ 13-14 or Δ PDZ construct cDNAs under the control of the C. elegans nlg-1 promoter. The Δ 13-16 and Δ 15-16 mutant constructs failed to rescue this behavioral deficit. Taken together, these results suggest that the alternatively spliced exons 13 and 14, and the PDZ binding motif are dispensable for NLG-1 localization and function in C. elegans, at least in our assays. In contrast, the intracellular potion of the protein encoded by exons 15 and 16 is required for NLG-1 function.
Figure 35. Deletion analysis of NLG-1: spontaneous reversal
Animals were removed from food and monitored for spontaneous reversal of direction. Each animal was observed for 3 minutes; each data bar represents the mean of 25 animals ± standard deviation. Transgenic expression of a Δ 13-14 or Δ PDZ construct rescued the spontaneous reversal defect while expression of a Δ 13-16 or Δ 15-16 did not. (***) indicates a statistically significant difference (P < .0001) between nlg-1 and wild-type, Δ 13-16 and wild type or Δ 15-16 and wild type. Δ 13-14 and Δ PDZ are not different than wildtype.
Figure 36. Deletion analysis of NLG-1: thermotaxis
Animals were grown at 20°C and placed on a thermal gradient as described in Methods. Transgenic expression of a Δ 13-14 NLG-1::YFP or Δ PDZ NLG-1::YFP fusion protein rescued the thermotaxis defect, while expression of a Δ 13-16 NLG-1::YFP or Δ 15-16 NLG-1::YFP fusion protein did not. Each data point represents the mean of six trials of ~50 animals each ± standard deviation.
Figure 37.Deletion analysis of NLG-1: Approach-Avoidance
Approach-Avoidance assays were performed as described in Methods and Figure 19. Transgenic expression of a Δ 13-14 NLG-1::YFP or Δ PDZ NLG-1::YFP fusion protein rescued the approach-avoidance defect, while transgenic expression of a Δ 13-16 NLG- 1::YFP or Δ 15-16 NLG-1::YFP fusion protein did not. Each bar represents the mean of six trials of ~50 animals each ± standard deviation. (***) indicates a statistically significant difference (P ≤ .0001) between nlg-1 and wild-type, Δ 13-16 and wild type or Δ 15-16 and wild type. Δ 13-14 and Δ PDZ are not different than wildtype.
Mutagenesis of putative PKC and Mek/Jnk binding sites
Mammalian data demonstrates that the neuroligin/neurexin interaction initiates a signaling event; however, the signaling mechanism is unknown. In an effort to determine whether phosphorylation plays a role in the intracellular signaling, localization, or functioning of C. elegans neuroligin, we identified several consensus sequences in the NLG-1 C-terminus for PKC phosphorylation sites and performed site-directed mutagenesis on the NLG-1::YFP construct. We deleted amino acids 796-802 to remove two of the potential PKC sites. We performed this mutagenesis in RM#1018p which lacks alternative exons 13 and 14. This deletion did not affect the localization or function of the NLG-1::YFP fusion protein. Δ796-802 NLG-1::YFP localized to synaptic regions, and we observed discrete puncta in the dorsal nerve cord as well as the sublateral nerve cords. Furthermore, animals carrying this transgene showed rescue for all of the nlg-1 mutant phenotypes that we assayed (spontaneous reversal, octanol avoidance and thermotaxis, data not shown).
Figure 38 Diagram of a deletion eliminating two potential PKC sites
Since we had observed an oxidative stress phenotype in nlg-1 mutants, we evaluated stress pathways that might be affected by the loss of NLG-1. We identified an LAL motif, which is the consensus Mek/Jnk kinase binding site. To determine whether the LAL motif is a Mek/Jnk kinase binding site in C. elegans NLG-1, or if binding to Mek/Jnk kinases plays a role in the intracellular signaling, localization, or function of neuroligin, we performed site-directed mutagenesis on the neuroligin fusion protein, and changed amino acids 819-821 (LAL) to AAA. We found that this mutation did not affect the localization or function of the NLG-1::YFP fusion protein. The LAL→AAA NLG‑1::YFP fusion protein was localized to synaptic regions, and we observed discrete puncta in the dorsal and sublateral nerve cords. Furthermore, the LAL→AAA NLG-1::YFP fusion protein rescued all of the nlg-1 mutant phenotypes that we assayed (spontaneous reversal, octanol avoidance and thermotaxis, data not shown).
RNAi screen to identify putative localization factors
When the current study was initiated, neuroligin was thought to function as a “master switch” for synaptogenesis. Researchers in the field believed that the presence of neuroligin alone was sufficient, and perhaps even necessary, to induce synapse formation (Scheiffele et al., 2000, Levinson et al., 2005, Prange et al., 2004, Fu et al., 2003). If the presence of neuroligin by itself can dictate the location of a synapse, what determines the localization of neuroligin? To address this question, we undertook an RNAi screen for genes required for the localization of neuroligin in C. elegans. The introduction of double-stranded RNA into C. elegans by feeding them Escherichia coli expressing target-gene dsRNA (Timmons et al., 2001) results in rapid, targeted, potent and systemic inactivation of an endogenous gene with corresponding sequence. The Ahringer lab constructed an RNAi feeding library of 16,757 bacterial strains (targeting ~86% of predicted genes) for use in genome-wide RNAi screening in C. elegans (Fraser et al., 2000, Kamath and Ahringer, 2003, Kamath et al., 2003). We took advantage of this library (gift of Dr. Robert Barstead) to knock down a list of candidate genes. Rather than perform a whole-genome screen, we chose to focus on all proteins that are predicted to have PDZ domains, some synaptic scaffolding molecules, motor proteins and proteins that are known to affect localization of synaptic components (Table 5). We introduced an integrated Pnlg-1::NLG-1::YFP transgene (mdIs168) into an rrf-3 mutant background. In C. elegans, neurons are resistant to RNAi in a wild-type genetic background (Timmons et al., 2001); rrf-3 mutations result in enhanced sensitivity to RNAi in many cell types, including neurons (Simmer et al., 2002). mdIs168; rrf-3 animals were cultured on plates seeded with bacteria expressing double-stranded RNAi corresponding to each of the 75 genes selected (see Methods). After several generations, worms were fixed and stained, using antibodies to GFP and the active zone protein RIM. Worms were then examined under a compound microscope for mislocalization of the NLG-1::YFP relative to the active zone marker. Although this screen was highly labor intensive, I was able to identify two RNAi clones, targeting two genes, which reproducibly disrupted the localization of neuroligin.
The first gene, par-3, encodes a PDZ domain-containing protein that is homologous to mammalian atypical PKC isotype-specific interacting protein (ASIP) and Drosophila Bazooka. The PAR-3 protein is required for the polarization of the embryo along the anterior-posterior axis (Cheng et al., 1995). Mutations in the par-3 gene result in maternal-effect lethality, making this gene difficult to work with.
The second gene, unc-116, encodes a kinesin-1 heavy chain. The UNC‑116 protein functions as an anterograde microtubule-based motor that is required for transport and localization of dense core vesicle components (Bowman et al., 2000, Sakamoto et al., 2005, Byrd et al., 2001). The idea of a motor protein being required to localize neuroligin was appealing. Based on this idea, we tested whether the UNC-104/KIF1 protein also has a role in neuroligin localization. UNC-104/KIF1 is also a kinesin-like molecule that is involved in synaptic vesicle trafficking (Otsuka et al., 1991). However, RNAi knockdown of unc-104 appeared to have no effect on NLG‑1::YFP localization (but see below).
A potential limitation of our approach is that the neuroligin promoter is expressed in ~45 neurons, making it difficult to examine pre- or postsynaptic regions in specific neurons. In addition, the neuroligin promoter is expressed in muscle, and we would therefore not be able to see the difference between pre-synaptic and post-synaptic neuroligin at NMJs. Therefore, we expressed NLG::YFP under control of the itr-1B promoter, which, beginning at the L4 stage, is expressed in a single motor neuron, DA9, in the tail of the animal. The DA9 neuron has a simple polarized anatomy; the presynaptic region is part of the dorsal nerve cord, while the postsynaptic region (and the neuronal cell body) is part of the ventral nerve cord. This neuron normally expresses NLG-1.
We examined the role of the UNC-116 and UNC-104 proteins in localizing neuroligin by introducing the Pitr-1B::NLG-1::YFP transgene into unc-104 and unc-116 mutant backgrounds. In the unc-116 mutant background, NLG::YFP could not be observed in either the dorsal or ventral nerve cord. We conclude that UNC-116 is required for both the pre- and postsynaptic localization of neuroligin. In unc-104 background, NLG::YFP is absent from the dorsal nerve cord. We conclude that UNC-104/KIF1 is also required for the presynaptic localization of neuroligin.
Mutagenesis and screen to identify putative localization mutants
The RNAi screen to identify putative localization factors had several important limitations. First, the set of targeted genes was chosen to emphasize proteins with specific structural domains or biological roles. Clearly, it is not possible to anticipate all possible relevant genes or proteins. Secondly, the fixing, staining and imaging of large numbers of treated worms was very labor intensive. To address these limitations, we undertook a forward genetic screen based on a simple premise: we hypothesized that mutants with defects in neuroligin localization would have nlg-1-like mutant phenotypes. Octanol avoidance (see Methods: chemotaxis assays) was used as a primary screen. Worms that failed to avoid octanol were then scored for spontaneous reversal (see Methods: spontaneous reversal) as a secondary screen. Worms who met both criteria were cultured, and the offspring were fixed and stained with anti-GFP and anti‑RIM antibodies. We screened ~3,000 genomes, and identified nine mutants that met both of these screening criteria. Unfortunately, none of these animals had obvious neuroligin localization defects and, therefore, were not analyzed further.